Vitrification is a process when a liquid or semi-liquid biological sample becomes highly viscous, avoiding intracellular and intercellular ice formation and thus, increasing chances for survival; an amorphous vitreous (glassy) phase is formed. This glassy state may be achieved in most liquids by means of very fast cooling. Thus, for example, pure water vitrification is achieved at the cooling rate of about 108 K/min. Utilization of cryoprotective agents (CPAs) significanty increases these extremely high cooling rates to rapid (higher 10,000° C Min) or ultra-rapid (ultra-fast) cooling rates (above 10,000° C/Min but below 100,000° C/Min). This method is very attractive for cryopreservation of biological samples. High concentrations of permeable CPAs must be used for the most widely used methods of equilibrium (slow) and quasi-kinetic vitrification with relatively more rapid rates of cooling, including ultra-rapid (higher than 20,000° C/Min but below 50,000° C/Min) vitrification. Those CPAs, used in equilibrium or quasi-kinetic vitrification, which comprise, but are not limited to, glycerol, [Katkov et al., 2012], can substantially damage the cells even without vitrification due to either osmotic damage or specific chemical toxicity [Katkov & Pogorelov, 2007; Katkov, 2011]. The Leidenfrost effect (LFE) is a vapor film formation (film boiling) on the site of the contact between the coolant/heater and heated/cooled sample. In the former case, there is heating LFE, which can be observed when a droplet of water is placed on overheated metallic pan. In the latter case, there is cryogenic LFE, when a liquid coolant boils and forms a vapor coating around the cooling sample (which can be both liquid or solid). It is the major factor that substantially impedes the rate of the surface cooling. All the devices referenced above where the samples are immersed into liquid nitrogen or other coolant, have substantially lower cooling rates than as claimed because of the cryogenic LFE.
It would therefore be advantageous to reduce the LFE effect, to improve the efficiency of cooling, and to simultaneously reduce the need for toxic CP As. Some embodiments of the present invention can achieve these goals with hyper-fast cooling rates (50,000° C/Min and higher) by reducing the LFE effect, and by totally eliminating or substantially decrease the use of potentially toxic permeable CPAs mentioned above.
Another goal of this invention is to insure hyper-fast re-warming of the vitrified samples and avoid the re-crystallization or the growth of new crystals during warming of the kinetically vitrified sample